(+)-cc-1065 (C. G. Chidester et. al., J. Amer. Chem. Soc., 103, 7629 (1981)) and the duocarmycins (Duocarmycin SA: M. Ichimura et al., J. Antibiot., 1990, 43, 1037; M. Ichimura et al., J. Antibiot., 1991, 44, 1045; Duocarmycin A, B.sub.1, B.sub.2, C.sub.1 and C.sub.2 : M. Ichimura et al., J. Antiobiot., 1988, 41, 1285; I. Takehashi et. al., J. Antiobiot., 1988, 41, 1915; T. Yasuzawa et. al., Chem. Pharm. Bull., 1988, 36, 3728; T. Ogawa et al., J. Antiobiot., 1989, 42, 1299; Pyrindamycin A and B: Ohba et al., J. Antiobiot., 1988, 41, 1515; S. Ishii et al., J. Antiobiot., 1989, 42, 1713) constitute exceptionally potent naturally occurring antitumor antibiotics, isolated from Streptomyces species that heve been shown to be related through their common participation in a characteristic minor grrove adenine N3 alkylation duplex DNA ( M. A. Warpehoski et. el., Chem. Res. Toxicol., 1988, 1, 315; L. Hurley et al., Acc. Chem. Res., 1986, 19, 230; D. L. Boger in Advances in Heterocylic Natural Product Synthesis, Vol. 2, W. H. Pearson; Ed., JAI Press, Greenwich, 1992, 1-188; D. L. Boger, Chemtracts: Org. Chem., 1991, 4, 329; R. S. Coleman and D. S. Boger in Studies in Natural Products Chemistry; Atyta-ur-Rahman, Ed., Elsevier, Amsterdam, 1989, Vol.3, 301; V. H. Rawal et. el., Heterocycl., 1987, 25, 701).
Since the disclosure of the structure of (+)-CC-1065, the agent and structural analogs have been the subject of continued synthetic as well as biological studies. Numerous synthetic analogs of CC-1065 and the duocarmycins have been synthesized incorporating deep-seated changes in the alkylating subunit with the intent of determining the fundamental structural features contributing to the polynucleootide recognition and funtional reactivity (D. L. Boger et al., Pure & Appl. Chem., 1993, 65, 1123; D. L. Boger et al., J. Amer. Chem. Coc., 992, 114, 9318; J. Amer. Chem., Soc., 1992, 114, 5487; D. L. Boger et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 1431; D. L. Boger et al., Bioorg. & Med. Chem. Letts, 1991, 1, 55; J. Amer. Chem. Soc., 1992, 112, 5230; J. Org. Chem., 1990, 55, 5823; J. Org. Chem., 1989, 54, 1238; M. A. Warpehoski et al., J. Med. Chem., 1988, 31, 590). The cytotoxic potency of CC-1065 has been correlated with the alkylating activity and DNA-binding or DNA-interchelating activity of CC-1065. The two activities reside in two separate pads of the molecule. The alkylating activity is contained in the CPI unit A (cyclopropapyrroloindole unit) and the DNA-binding in the two subunits of the molecule.
CC-1065 is 100 to 1000-fold more cytotoxic than conventional cancer chemotherapeutic agents such as duanorubicin, vincristine and methotrexate (B. K. Bhuyan et al., Cancer Res., 1982, 42, 3532). Although CC-1065 showed moderate anti-tumor activity in vivo, it was not evaluated clinically because it caused delayed deaths in mice at therapeutic doses (J. P. McGovren et al., J. Antibiot., 1984, 37, 63; V. L. Reynolds et al., J. Antibiot., 1986, 39, 319). The synthesis of new analogs of CC-1065 that retain the high in vitro cytotoxicity of the parent drug without causing delayed lethality in mice have been reported (M. A. Warpehoski et al., J. Med. Chem., 1988, 31, 590-603). Adozelesin (U-73975) is one of many CC-1065 analogues with excellent broad-spectrum anti-tumor activity in vivo without causing delayed deaths (L.H. Li et al., Invest. New Drugs, 1991, 9, 137).
The high toxicity of CC-1065 and the related duocarmycins has prompted the synthesis of prodrug analogues. Ester and urethane analogs of the CPI phenol of the chloromethyl precursor to the cyclopropyl function typified by Carzelesin (U-80244) (FIG. 4) have proved to be more efficacious in vivo than the parent CPI drugs (L. H. Li et al., Cancer Res., 1992, 52, 4904). Hydrolysis of such esters or urethanes to reform the chloromethyl phenol compound must occur prior to the ring-closure reaction, in order to produce the DNA-reactive cyclopropyl keto compound. A similar prodrug strategy was recently reported for Duocarmycin B2 (H. Ogasawara et al., in Proceedings of the 85th Annual Meeting of the American Association for Cancer Research, Abstract 2325, p390, 1994).
The use of tumor specific antibodies to the targeted delivery of enzymes to the surface to tumor cells in combination with specific enzyme activated prodrugs has been employed in an effort to improve the therapeutic efficacy of a number of cytotoxic drugs. Prodrugs derived from etoposide and mitomycin C in combination with alkaline phosphatase antibody conjugates (P. D. Senter et al., Proc. Natl. Acad. Sci. USA. 1988, 85, 4842; P. D. Senter et al., Cancer Res. 1989, 49, 5789; H. J. Haisma et al., Cancer Immunol. Immunotherap., 1992, 34, 343); bifunctional alkylating agents and carboxypeptidase G2 (C. J. Springer et al., Eur. J. Cancer, 1991, 27 (11), 1361) and bacterial .beta.-glucuronidase (S. -M. Wang et al., Cancer Res., 1992, 52, 4484); vinca alkaloids and .beta.-lactamase (D. L. Meyer et al., Cancer Res., 1993, 53, 3956); and methoxtrexate and carboxypeptidase A (E. Haenseler et al., Biochemistry, 1992, 31, 891) have been reported in the recent literature. This antibody directed enzyme prodrug therapy, "ADEPT" approach has demonstrated efficacy both `in vivo` and in `in vivo` animal model studies. A glucuronide spacer prodrug derivative of doxorubicin has been employed together with a hymanized carcinoembryonic antigen-specific recombinant variable region fused to human .beta.-glucuronidase in an `in vivo` study with nude mice bearing human CEA expressing tumor zenografts. The two component fusion protein/prodrug system showed therapeutic effects superior to those of conventional chemotherapy (K. Bosslet et al., Cancer Res., 1994, 54, 2151).
Additionally, mAb-enzyme conjugates have been used for the site-specific formation of cytotoxic agents. The principle advantage of this approach compared to those involving the direct attachment of a cytotoxic agent to a mAb is that the targeted enzyme can greatly amplify the number of drug molecules delivered to each tumor cell. Furthermore, the cytotoxic effect may not be restricted to only those tumor cells that have bound the conjugate, since the drug is released extracellularly and may be able to migrate to neighboring tumor cells. The principle disadvantage lies in the approach's inherent complexity, since two separate agents and an appropriate time interval between their respective administrations are required for therapeutic efficacy.
Mab-enzyme conjugates can release a vast array of cytotoxins ranging from clinically approved anticancer drugs to highly potent agents that would have little chance for success in the clinic if given systemically. Several groups have demostrated that significant in vitro and in vivo antitumor activities can be obtained using this approach, and clinical studies are now underway. Further refinements in the technology, such as the use of recombinant fusion proteins comprises of relatively nonimmunogenic components, the development of prodrugs with optimal pharmacological distributions and minimal toxicities, and strategies to achieve high tumor to normal tissue conjugate ratios, may provide the basis for making this approach clinically useful.
The use of enzyme-acceptor and enzyme-donor polypeptides in enzyme complementation assays is described in U.S. Pat. No. 4,708,929. The prior art is silent on the use of this technology in the generation of cytotoxic agents by targeted enzymes.